Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15 Minutes of Antibiotic Exposure

Rapid antimicrobial susceptibility testing (AST) would decrease misuse and overuse of antibiotics. The “holy grail” of AST is a phenotype‐based test that can be performed within a doctor visit. Such a test requires the ability to determine a pathogen's susceptibility after only a short antibiotic exposure. Herein, digital PCR (dPCR) was employed to test whether measuring DNA replication of the target pathogen through digital single‐molecule counting would shorten the required time of antibiotic exposure. Partitioning bacterial chromosomal DNA into many small volumes during dPCR enabled AST results after short exposure times by 1) precise quantification and 2) a measurement of how antibiotics affect the states of macromolecular assembly of bacterial chromosomes. This digital AST (dAST) determined susceptibility of clinical isolates from urinary tract infections (UTIs) after 15 min of exposure for all four antibiotic classes relevant to UTIs. This work lays the foundation to develop a rapid, point‐of‐care AST and strengthen global antibiotic stewardship.     

A self-loading microfluidic device for determining the minimum inhibitory concentration of antibiotics

This article describes a portable microfluidic technology for determining the minimum inhibitory concentration (MIC) of antibiotics against bacteria. The microfluidic platform consists of a set of chambers molded in poly(dimethylsiloxane) (PDMS) that are preloaded with antibiotic, dried, and reversibly sealed to a second layer of PDMS containing channels that connect the chambers. The assembled device is degassed via vacuum prior to its use, and the absorption of gas by PDMS provides the mechanism for actuating and metering the flow of fluid in the microfluidic channels and chambers. During the operation of the device, degas driven flow introduces a suspension of bacterial cells, dissolves the antibiotic, and isolates cells in individual chambers without cross contamination. The growth of bacteria in the chambers in the presence of a pH indicator produces a colorimetric change that can be detected visually using ambient light. Using this device we measured the MIC of vancomycin, tetracycline, and kanamycin against Enterococcus faecalis 1131, Proteus mirabilis HI4320, Klebsiella pneumoniae, and Escherichia coli MG1655 and report values that are comparable to standard liquid broth dilution measurements. The device provides a simple method for MIC determination of individual antibiotics against human pathogens that will have applications for clinical and point-of-care medicine. Importantly, this device is designed around simplicity: it requires a single pipetting step to introduce the sample, no additional components or external equipment for its operation, and provides a straightforward visual measurement of cell growth. As the device introduces a novel approach for filling and isolating dead-end microfluidic chambers that does not require valves and actuators, this technology should find applications in other portable assays and devices.     

Detecting bacteria and determining their susceptibility to antibiotics by stochastic confinement in nanoliter droplets using plug-based microfluidics

This article describes plug-based microfluidic technology that enables rapid detection and drug susceptibility screening of bacteria in samples, including complex biological matrices, without pre-incubation. Unlike conventional bacterial culture and detection methods, which rely on incubation of a sample to increase the concentration of bacteria to detectable levels, this method confines individual bacteria into droplets nanoliters in volume. When single cells are confined into plugs of small volume such that the loading is less than one bacterium per plug, the detection time is proportional to plug volume. Confinement increases cell density and allows released molecules to accumulate around the cell, eliminating the pre-incubation step and reducing the time required to detect the bacteria. We refer to this approach as 'stochastic confinement'. Using the microfluidic hybrid method, this technology was used to determine the antibiogram - or chart of antibiotic sensitivity - of methicillin-resistant Staphylococcus aureus (MRSA) to many antibiotics in a single experiment and to measure the minimal inhibitory concentration (MIC) of the drug cefoxitin (CFX) against this strain. In addition, this technology was used to distinguish between sensitive and resistant strains of S. aureus in samples of human blood plasma. High-throughput microfluidic techniques combined with single-cell measurements also enable multiple tests to be performed simultaneously on a single sample containing bacteria. This technology may provide a method of rapid and effective patient-specific treatment of bacterial infections and could be extended to a variety of applications that require multiple functional tests of bacterial samples on reduced timescales.     

Increasing antibiotic activity against a multidrug-resistant Acinetobacter spp by essential oils of Citrus limon and Cinnamomum zeylanicum

The genus Acinetobacter has gained importance in recent years due to involvement in serious infections and antimicrobial resistance. Many plants have been evaluated not only for direct antimicrobial activity, but also as resistance modifying agents. The Essential oil of Citrus limon (EOCL) addition at 156.25?μgmL(-1) (MIC/8) sub-inhibitory concentration in the growth medium led to MIC decrease for amikacin, imipenem and meropenem. The Essential oil of Cinnamomum zeylanicum (EOCZ) addition at 78.125?μg?mL(-1) (MIC/8) sub-inhibitory concentrations in the growth medium caused drastic MIC reduction of amikacin. Results of combining antibiotics and essential oils had shown us a synergistic effect with both essential oils/amikacin combinations. An additive effect was observed with the combinations of both essential oils and gentamicin. The results of this study suggest that essential oil of C. limon and C. zeylanicum may suppress the growth of Acinetobacter species and could be a source of metabolites with antibacterial modifying activity.     

Preliminary phytochemical screening and in vitro anti-helicobacter pylori activity of extracts of the stem bark of Bridelia micrantha (Hochst., Baill., Euphorbiaceae)

Helicobacter pylori is a major risk factor for gastritis, ulcers and gastric cancer. This study was aimed to determine the antimicrobial activity of the stem bark of Bridelia. micrantha on H. pylori isolated in South Africa. Extracts and clarithromycin were tested against 31 clinical strains, including a standard strain (NCTC 11638) of H. pylori, by measuring the diameters of the corresponding inhibition zones, followed by determination of the Minimum Inhibitory Concentration (MIC) (using metronidazole, and amoxicillin as control antibiotics) and the rate of kill. Preliminary phytochemical screening was also done. Inhibition zone diameters which ranged from 0-23 mm were observed for all five of the extracts and 0-35 mm for clarithromycin. Marked susceptibility of strains (100%) was noted for the acetone extract (P < 0.05), followed by ethyl acetate extract (93.5%). The MIC?? values ranged from 0.0048 to 0.156 mg/mL for the ethyl acetate extract and 0.0048 to 0.313 mg/mL for the acetone extract. The MIC?? values ranged from 0.0048 to 2.5 mg/mL for the ethyl acetate extract and 0.078 to > 0.625 mg/mL for the acetone extract, respectively. Insignificant statistical difference in potency was observed when comparing the crude ethyl acetate extract to metronidazole and amoxicillin (P > 0.05). Complete killing of strain PE430C by the ethyl acetate extract was observed at 0.1 mg/mL (2 × MIC) and 0.2 mg/mL (4 × MIC) at 66 and 72 h. For strain PE369C, 100% killing was observed at 0.1 mg/mL (2 × MIC) in 66 and 72 h. The ethyl acetate extract could thus be a potential source of lead molecules for the design of new anti-Helicobacter pylori therapies as this study further confirmed the presence of phytochemicals including alkaloids, flavonoids, steroids, tannins and saponins.     

Human neural stem cell growth and differentiation in a gradient-generating microfluidic device

This paper describes a gradient-generating microfluidic platform for optimizing proliferation and differentiation of neural stem cells (NSCs) in culture. Microfluidic technology has great potential to improve stem cell (SC) cultures, whose promise in cell-based therapies is limited by the inability to precisely control their behavior in culture. Compared to traditional culture tools, microfluidic platforms should provide much greater control over cell microenvironment and rapid optimization of media composition using relatively small numbers of cells. Our platform exposes cells to a concentration gradient of growth factors under continuous flow, thus minimizing autocrine and paracrine signaling. Human NSCs (hNSCs) from the developing cerebral cortex were cultured for more than 1 week in the microfluidic device while constantly exposed to a continuous gradient of a growth factor (GF) mixture containing epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF). Proliferation and differentiation of NSCs into astrocytes were monitored by time-lapse microscopy and immunocytochemistry. The NSCs remained healthy throughout the entire culture period, and importantly, proliferated and differentiated in a graded and proportional fashion that varied directly with GF concentration. These concentration-dependent cellular responses were quantitatively similar to those measured in control chambers built into the device and in parallel cultures using traditional 6-well plates. This gradient-generating microfluidic platform should be useful for a wide range of basic and applied studies on cultured cells, including SCs.     

Parallel analysis of antimicrobial activities in microbial community by SSCP based on CE

Conventional antimicrobial activity analyses such as the broth dilution method and disk diffusion test are considerably demanding processes for new antimicrobial agent discovery and sensitive diagnosis of infectious diseases. Here, we developed a new antimicrobial activity analysis system using CE-based SSCP (CE-SSCP) combined with 16S rRNA gene-specific PCR (PCR/CE-SSCP). Using this method, the population change in the microbial community in response to specific antimicrobial agents could be quantified with a high sensitivity and accuracy from a small sample amount. Using a mixture of microorganisms comprising Escherichia coli, Corynebacterium glutamicum, Acinetobacter calcoaceticus, and Staphylococcus aureus as a model system, the linear correlation between the genomic DNA concentrations and peak areas in 16S rRNA gene-specific PCR/CE-SSCP was determined; consequently, quantification of cell concentrations could be demonstrated using this method. Compared to the minimum inhibitory concentration (MIC) values from the conventional broth dilution method, this new system provided almost the same MIC values for popular antimicrobial agents such as kanamycin, spectinomycin, and streptomycin. The results demonstrated that the newly developed method can be a substitute for the conventional antimicrobial analysis method and highlighted its high potential in the areas of new antimicrobial agent discovery and clinical diagnosis.     

Integrated microfluidic platform for rapid antimicrobial susceptibility testing and bacterial growth analysis using bead-based biosensor via fluorescence imaging

The paper describes a droplet-based microfluidic method for phenotypic-based antimicrobial susceptibility testing (AST). In particular, this micro-droplet-based phenotypic assay evaluates susceptibility of different bacterial strains towards antibiotics by tracking effects on individual bacterial cells, including changes in bacterial cell number and morphology. The platform was validated by applying the method to test the responses of E. coli ATCC 25922 and 6937 (a clinical isolate), in spiked urine samples at a concentration of 5 × 10⁴ cfu mL^?1, to the antibiotics ceftazidime and levofloxacin. Both E. coli strains showed dose-dependent inhibition of bacterial replication and morphological alteration. These correlated well with minimal inhibitory concentrations determined by the reference broth microdilution method. Discrete bacterial divisions and morphological changes were observed within 20 min of on-chip incubation, demonstrating performance of rapid AST directly on urine samples. As proof-of-concept, specific bead-based biosensors were tested for capture and detection of E. coli for on-bead proliferation. The method has the attractive feature of allowing the detection of at least one bacterium per bead in less than 30 min. It can potentially be used to isolate a specific bacterial strain directly from patient urine samples for AST monitoring.

Open image in new window

Graphical Abstract (A) Schematic of the droplet microfluidic chip for bacterial detection and Antibiotic Susceptibility Testing (AST); (B) Time lapse proliferation images of green fluorescent protein expressing E. coli in droplets. (C) Bacterial proliferation on the bead-based sensor.

     

Design of antibacterial peptide-like conjugated molecule with broad spectrum antimicrobial ability

Increasing multidrug-resistant(MDR) superbugs emerge worldwide causing a public health crisis. Consequently, it is urgent to find new antibiotics with efficient broad-spectrum antimicrobial activity. By virtue of versatility in molecular design, a new peptide-like cell membrane-broken molecule, oligo-(7,7′-bifluoren-benzo[c][1,2,5]thiadiazole)(OFBT) possessing a conjugated backbone and eight pendant guanidyl moieties was designed and synthesized. OFBT exhibits favorable broad-spectrum of antimicrobial activity to pathogens including Gram-negative and Gram-positive bacteria, and fungi with the minimum inhibitory concentration(MIC)below 3.0 μM. Moreover, OFBT exhibits high selectivity for pathogens over human cells to make it a promising broad spectrum antimicrobial agent.     

Microfluidic system for simultaneous optical measurement of platelet aggregation at multiple shear rates in whole blood

Thrombosis is the pathological formation of platelet aggregates which occlude blood flow causing stroke and heart attack-the leading causes of death in developed nations. Instrumentation for diagnosing and exploring treatments for pathological platelet aggregation thus has the potential for major clinical impact. Most current thrombosis methods focus on single flow conditions, non-occlusive platelet adhesion, or low shear rates and so are limited in their application to comparative studies involving multiple, pathological test conditions (e.g., shear rate, stenotic geometries that mimic arteries, and rapid platelet accumulation to occlusion). The field could benefit from a low volume, high throughput, short analysis time, and low cost system while minimizing sample handling. We report on the design, fabrication, testing, and application of a microfluidic device and associated optical system for simultaneous measurement of platelet aggregation at multiple initial shear rates within four stenotic channels in label-free whole blood. Following computational design, requisite shear rates were achieved in the device by micro- surface milling a mold and subsequent PDMS casting. We applied the microfluidic system to measure platelet aggregation in whole porcine blood for shear rates spanning physiological to pathological flow conditions (500-13000 s(-1)). Real-time, non-contact, label-free, microscope-free measurements of platelet aggregation were acquired using an optical system comprising a 650 nm diode laser and a linear CCD. We observed fully occlusive platelet aggregation in less than 20 min above a threshold initial shear rate of 4000 s(-1), and no occlusive platelet aggregation below 1500 s(-1) (N = 86 trials). Accumulation of thrombus was consistent between laser intensity, light microscopy, histology, and mass flow rate measurements. The amount of blood volumes producing occlusion were dependent on shear rate. Times to occlusion were not found to be dependent on shear rate above the threshold level of 4000 s(-1). This microfluidic system enables measurement of the entire process of occlusive platelet thrombosis in whole, unlabeled blood, in vitro, at multiple shear rates. Such a system may be useful as a point-of-care diagnostic tool for studying anti-platelet therapies in individual blood samples from high-risk patients.     

Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution

We have developed a microfluidic platform that enables, in one experiment, monitoring of signaling events spanning multiple time-scales and cellular locations through seamless integration of cell culture, stimulation and preparation with downstream analysis. A combination of two single-cell resolution techniques-on-chip multi-color flow cytometry and fluorescence imaging provides multiplexed and orthogonal data on cellular events. Automated, microfluidic operation allows quantitatively- and temporally-precise dosing leading to fine time-resolution and improved reproducibility of measurements. The platform was used to profile the toll-like receptor (TLR4) pathway in macrophages challenged with lipopolysaccharide (LPS)-beginning with TLR4 receptor activation by LPS, through intracellular MAPK signaling, RelA/p65 translocation in real time, to TNF-α cytokine production, all in one small macrophage population (< 5000 cells) while using minute reagent volume (540 nL/condition). The platform is easily adaptable to many cell types including primary cells and provides a generic platform for profiling signaling pathways.     

Antimicrobial and hemolytic activities of copolymers with cationic and hydrophobic groups: a comparison of block and random copolymers

Random and diblock copolymers of 2-(N,N-dimethylamino)ethyl methacrylate and butyl methacrylate are prepared by ATRP. As mimics of cationic antimicrobial peptides, the random and diblock copolymers show similar antimicrobial activities. In contrast, the diblock copolymers have much lower hemolytic activities than the random copolymers. The cell selectivity (HC(50)/MIC, where HC(50) is the concentration to lyse 50% of human red blood cells and MIC is the minimum concentration to inhibit bacterial growth) of the diblock copolymers are 150 to 27,500 times higher than that of random copolymers with similar compositions.     

Shrinky-Dink microfluidics: 3D polystyrene chips

We present a novel approach for the ultra-rapid direct patterning of complex three-dimensional, stacked polystyrene (PS) microfluidic chips. By leveraging the inherent shrinkage properties of biaxially pre-stressed thermoplastic sheets, microfluidic channels become thinner and deeper upon heating. Design conception to fully functional chips can thus be completed within minutes.     

Antimicrobial activities of essential oil and methanol extract of Coriaria nepalensis

Plant extracts and products have been used for centuries in traditional medicine; for most of them, in addition to the scant scientific credibility, the chemical composition and spectrum of activity are yet to be explored. To put forward this effort and to identify novel antimicrobial agents, the inhibitory activities of methanolic extract and essential oil from Coriaria nepalensis against various microorganisms including pathogenic yeast, and Gram-positive and negative bacteria were evaluated. Chemical compositions of C. nepalensis methanolic extract and essential oil were analysed by gas chromatography-mass spectrometry. In vitro susceptibility tests against all the tested isolates were performed in terms of minimum inhibitory concentration (MIC), and well diffusion assay using standard protocols. All microorganisms tested were profoundly found susceptible to both the C. nepalensis extract and oil with MIC values of 1.3-2.1?mg?mL?1 (Gram-positive bacteria), 1.4-2.2?mg?mL?1 (Gram-negative bacteria) and 0.9-1.6?mg?mL?1 (yeasts). The extent of inhibition was shown more by methanolic extract than by essential oil. This study is the first to report the antimicrobial activity of extracts obtained from the C. nepalensis. It can be concluded that the observed antimicrobial characteristics of C. nepalensis indicate that it might be a promising antimicrobial agent.     

In vitro antibacterial activity of medicinal plant extracts against Escherichia coli strains from human clinical specimens and interactions with antimicrobial drugs

The biological properties of medicinal plants have been documented worldwide for many centuries. We aimed to evaluate interactions between crude extracts from Psidium guajava, Zingiber officinale, Cymbopogon citratus, Caryophyllus aromaticus, Mikania glomerata and Allium sativum samples and antimicrobial drugs against Escherichia coli strains. The susceptibility test performed was disc diffusion, and crude extracts were diluted (%v/v) into Müller-Hinton agar (MHA) at one quarter of the minimal inhibitory concentration for 90% (MIC(90%)) of E. coli strains found previously. Synergistic interactions were observed between C. citratus and polymyxin, and A. sativum extracts and gentamicin. The crude A. sativum extract was the only one that did not show any antagonism with the antimicrobial drugs. The results thus showed the potential use of these medicinal plants against E. coli strains, although antagonism with antimicrobial drugs is a negative aspect in the combined therapy of infectious diseases caused by E. coli.     

Novel Pyrazoline‐based Organometallic Compounds Containing Ferrocenyl and Quinoline units: Synthesis,Characterization and Microbial susceptibilities

Some novel pyrazoline‐based organometallic compounds were synthesized as new leads in antimicrobial chemotherapy. The structures of compounds were elucidated by different spectroscopic techniques and elemental analyses. All compounds were investigated for in vitro antimicrobial studies against fifteen ATTC bacterial and fungal strains. The microbial susceptibility of these compounds revealed that all the tested compounds gave good minimum inhibitory concentration (MIC) values against the tested organisms that are either similar or even better than the reference drugs amoxicillin and fluconazole, which gave MIC values 8‐64 μg/ml against bacterial and 64 μg/ml against fungal strains, respectively. Among all compounds, compound ( 4d ) 1‐(5‐(4‐chlorophenyl)‐3‐ferrocenyl‐4,5‐dihydropyrazol‐1‐yl)‐2‐quinolin‐8‐yloxy) ethanone, emerged out the most promising antimicrobial organometallic derivative with MIC values against all the strains ranging from 8‐32 μg/ml. Other compounds gave a range of MIC values between 16‐64 μg/ml against S. bovis, 16‐32 μg/ml against E. coli, and C. tropicalis except compound ( 4d) which gave MIC 8 μg/ml against S. bovis and E. coli, whereas 32 μg/ml against C. tropicalis. Collectively, these compounds gave a lower MIC value between 32‐64 μg/ml against both of the biofilm forming strains namely, P. aeruginosa and S. mutans. The results of microbial susceptibility concluded that these novel organometallic compounds are new leads in antimicrobial chemotherapy and can be very useful for further optimization work on microbial chemotherapy.     

Monitoring induced gene expression of single cells in a multilayer microchip

We present a microfluidic system that facilitates long-term measurements of single cell response to external stimuli. The difficulty of addressing cells individually was overcome by using a two-layer microfluidic device. The top layer is designed for trapping and culturing of cells while the bottom layer is employed for supplying chemical compounds that can be transported towards the cells in defined concentrations and temporal sequences. A porous polyester membrane that supports transport and diffusion of compounds from below separates the microchannels of both layers. The performance and potential of the device are demonstrated using human embryonic kidney cells (HEK293) transfected with an inducible gene expression system. Expression of a fluorescent protein (ZsGreen1-DR) is observed while varying the concentration and exposure time of the inducer tetracycline. The study reveals the heterogeneous response of the cells as well as average responses of tens of cells that are analyzed in parallel. The microfluidic platform enables systematic studies under defined conditions and is a valuable tool for general single cell studies to obtain insights into mechanisms and kinetics that are not accessible by conventional macroscopic methods.     

Multiplexing microelectrodes for dielectrophoretic manipulation and electrical impedance measurement of single particles and cells in a microfluidic device

This work presents a microfluidic device, which was patterned with (i) microstructures for hydrodynamic capture of single particles and cells, and (ii) multiplexing microelectrodes for selective release via negative dielectrophoretic (nDEP) forces and electrical impedance measurements of immobilized samples. Computational fluid dynamics (CFD) simulations were performed to investigate the fluidic profiles within the microchannels during the hydrodynamic capture of particles and evaluate the performance of single‐cell immobilization. Results showed uniform distributions of velocities and pressure differences across all eight trapping sites. The hydrodynamic net force and the nDEP force acting on a 6 μm sphere were calculated in a 3D model. Polystyrene beads with difference diameters (6, 8, and 10 μm) and budding yeast cells were employed to verify multiple functions of the microfluidic device, including reliable capture and selective nDEP‐release of particles or cells and sensitive electrical impedance measurements of immobilized samples. The size of immobilized beads and the number of captured yeast cells can be discriminated by analyzing impedance signals at 1 MHz. Results also demonstrated that yeast cells can be immobilized at single‐cell resolution by combining the hydrodynamic capture with impedance measurements and nDEP‐release of unwanted samples. Therefore, the microfluidic device integrated with multiplexing microelectrodes potentially offers a versatile, reliable, and precise platform for single‐cell analysis.     

Nanoparticles and surfaces presenting antifungal, antibacterial and antiviral properties

Here, we present new antimicrobial nanoparticles based on silica nanoparticles (SNPs) coated with a quaternary ammonium cationic surfactant, didodecyldimethylammonium bromide (DDAB). Depending on the initial concentration of DDAB, SNPs immobilize between 45 and 275 μg of DDAB per milligram of nanoparticle. For high concentrations of DDAB adsorbed to SNP, a bilayer is formed as confirmed by zeta potential measurements, thermogravimetry, and diffuse reflectance infrared Fourier transform (DRIFT) analyses. Interestingly, these nanoparticles have lower minimal inhibitory concentrations (MIC) against bacteria and fungi than soluble surfactant. The electrostatic interaction of the DDAB with the SNP is strong, since no measurable loss of antimicrobial activity was observed after suspension in aqueous solution for 60 days. We further show that the antimicrobial activity of the nanoparticle does not require the leaching of the surfactant from the surface of the NPs. The SNPs may be immobilized onto surfaces with different chemistry while maintaining their antimicrobial activity, in this case extended to a virucidal activity. The versatility, relative facility in preparation, low cost, and large antimicrobial activity of our platform makes it attractive as a coating for large surfaces.     

A microfluidic processor for gene expression profiling of single human embryonic stem cells

The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These 'cell population averaging' data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies.